It has been well known for many years that leguminous plants are able to fix nitrogen from atmospheric nitrogen due to a symbiotic relationship between the plant and bacteria which dwell in nodules formed in the roots of the plants. The symbiotic root nodule bacteria are now classified in at least several genera, e.g. Rhizobium, Bradyrhizobium, Sinorhizobium, and Azorhizobium. The three genera of nodule bacteria are characterized in part by the species of legume plant with which they are able to form the symbiotic nodulation relationship. Most of the bacteria which can nodulate soybean are Bradyrhizobium, although Sinorhizobium species can also nodulate soybean roots.
While significant research has been conducted on root nodulation bacteria in the hope of creating bacterial strains which will foster or improve the growth of legume plants cultivated agriculturally, the problem of increasing the effectiveness of inoculants of root nodule bacteria turns out to be a difficult one. In particular, for example, Bradyrhizobium japonicum strains now are extent in soils throughout North and South America in those regions in which there has been historic soybean production, even though the species was originally indigenous only to Asia. The existing wild strains are the progeny of bacterial strains originally inoculated into soybean fields but which have now evolved to survive in these soils and climates. The existence of these bacterial strains in the agricultural soil ecosystem is a mixed blessing. These strains of Bradyrhizobium extent in most cultivation areas compete with intentionally inoculated Bradyrhizobium strains for occupation of the nodules of soybean plants, and while the presently extent or native species may be inefficient fixers of nitrogen, they are often superbly adapted for competitive root nodulation in the particular environment or microenvironment in which they now exist and thrive. Accordingly, creating newly improved root modulation bacterial strains which are actually effective in the field as inoculants in increasing crop yields requires considerations of both increasing the effectiveness of the bacteria and also providing the improved or engineered bacteria with a mechanism by which they may compete effectively with bacterial strains now extent in most legume cultivation areas.
One of the characteristics of the nitrogen fixation process as performed by root nodulation bacteria is that a byproduct of the reaction is evolved hydrogen gas. For root nodule bacteria species which evolve hydrogen gas and release it into the atmosphere, a large amount of energy invested in the nitrogen fixation process is lost as the H.sub.2 gas is released into the atmosphere. However, it has been found that some diazotroph, or nitrogen fixing, bacteria do not evolve H.sub.2 under nitrogen fixation conditions. These bacteria were found to express an uptake hydrogenase enzyme which oxidized the hydrogen to protons and electrons. In the cases of some bacteria, the electron transport initiated by the hydrogenase results in an efficient energy conserving electron transport chain, which results in recovery of most of the energy that would otherwise be lost in hydrogen production.
The multi-gene for hydrogen uptake, designated HUP, was found to exist in several species of root nodulation bacteria. However, many other root nodule bacterial strains and species do not contain this capability, and thus are relatively wasteful in their energy utilization compared to species which have the capability of HUP expression. For example, there are no known HUP positive strains of Rhizobium etli or Bradyhizobium elkanii. It has also been found that strains of Bradyhizobium japonicum which are HUP positive appear to be scarce in agricultural soils.
It has been proposed that the HUP genes can be introduced into root nodule bacteria not natively possessing this phenotype to aid in their agricultural utility. U.S. Pat. No. 4,567,146 discusses one strategy for this approach. However, the potential introduction widespread agronomic potential of uptake hydrogenase phenotype in bacterial strains faces several hurdles. Among them is the fact that the HUP positive inoculant strains must be competitive for modulation with the endogenous strains now present in soils in crop growing areas. The HUP phenotype requires several genes and appears to be a competitive disadvantage in terms of metabolic burden to the bacteria. The second difficulty concerns the fact that there is an inherent instability in the expression of HUP genes in species which do not normally possess these genes. For example, Lambert et al., Appl. Environ. Microbiol., 53:422-428 (1987), were able to engineer hup expression in R. meliloti strains by conjugation of a cosmid clone containing the hup region, from a species of B. japonicum which contained the hup region. However, the expression was transient in root nodules because the lack of proper partitioning of the plasmid during cell division in the absence of selection pressure. Addition of tetracycline or other selection antibiotics to commercial inoculants to prevent improper partitioning is expensive, unlikely to be efficient, and could result in modification of animal or soil plant pathogens due to antibiotic resistance, and hence is not practical.
Of course, even if the HUP phenotype can be engineered into a strain of bacteria, there is still the competitiveness problem. One strategy which has been discussed for this problem is to engineer the root nodule bacteria with a toxin which is inhibitory to other root nodule bacterial. U.S. Pat. No. 5,183,759 describes root nodule bacteria engineered to produce trifolitoxin, one such toxin. Conferring a competitive advantage by adding trifolitoxin expression to a root nodule bacteria may not always be practical or effective. For example, it has been found difficult to express trifolitoxin production genes in Bradyrhizobium species. Also, ironically, it has been found that Bradyrhizobium species are several fold more resistant to trifolitoxin than species of Sinorhizobium fredii.
In considering the problem of engineering root nodulation bacteria for hydrogenase expression, another issue is the problem of strain by strain engineering of such bacteria. Now that the bacteria originally introduced as inoculants have evolved into discrete strains adapted to fit ecological conditions throughout agricultural regions it may be necessary for competitive reason to engineer different strains of bacteria for new traits for use in different agricultural regions of any given country or region. Accordingly, the ease with which a trait can be transferred among bacterial strains becomes a critical question in the practical use of engineered nitrogen fixing root nodule bacteria for use on field crops.